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Activation in Neutrophils BκAcute Lung Injury by Preventing NF-
C1P Attenuates Lipopolysaccharide-Induced
Gómez-Muñoz, Holger K. Eltzschig and Marco Idzko
Korcan Ayata, Madelon Hossfeld, Nicolas Ehrat, Antonio
Kristin Baudiß, Rodolfo de Paula Vieira, Sanja Cicko,
ol.1402681
http://www.jimmunol.org/content/early/2016/01/22/jimmun
published online 22 January 2016J Immunol
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is published twice each month byThe Journal of Immunology
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The Journal of Immunology
C1P Attenuates Lipopolysaccharide-Induced Acute Lung
Injury by Preventing NF-kB Activation in Neutrophils
Kristin Baudiß,* Rodolfo de Paula Vieira,* Sanja Cicko,* Korcan Ayata,*
Madelon Hossfeld,* Nicolas Ehrat,* Antonio Go
´mez-Mun
˜oz,
†
Holger K. Eltzschig,
‡
and
Marco Idzko*
Recently, ceramide-1-phosphate (C1P) has been shown to modulate acute inflammatory events. Acute lung injury (Arnalich et al. 2000.
Infect. Immun. 68: 1942–1945) is characterized by rapid alveolar injury, lung inflammation, induced cytokine production, neutrophil
accumulation, and vascular leakage leading to lung edema. The aim of this study was to investigate the role of C1P during LPS-
induced acute lung injury in mice. To evaluate the effect of C1P, we used a prophylactic and therapeutic LPS-induced ALI model in
C57BL/6 male mice. Our studies revealed that intrapulmonary application of C1P before (prophylactic) or 24 h after (therapeutic)
LPS instillation decreased neutrophil trafficking to the lung, proinflammatory cytokine levels in bronchoalveolar lavage, and alveolar
capillary leakage. Mechanistically, C1P inhibited the LPS-triggered NF-kB levels in lung tissue in vivo. In addition, ex vivo
experiments revealed that C1P also attenuates LPS-induced NF-kB phosphorylation and IL-8 production in human neutrophils.
These results indicate C1P playing a role in dampening LPS-induced acute lung inflammation and suggest that C1P could be a
valuable candidate for treatment of ALI. The Journal of Immunology, 2016, 196: 000–000.
Sphingolipids are structural molecules of eukaryotic cell
membranes. However, they act as second messenger
molecules in the regulation of cell homeostasis. Further-
more, sphingolipids were recently shown to contribute to the
regulation of inflammatory responses and could modify the de-
velopment and progression of human diseases (1, 2). A patho-
physiological role of sphingolipids is implicated in diabetes,
insulin resistance, neurodegenerative disorders, atherosclerosis,
and allergic airway inflammation (1–5). The key molecule in the
sphingolipid metabolism is ceramide, which regulates vital cel-
lular functions such as apoptosis, cell growth, and differentiation.
An important metabolite is ceramide-1-phosphate (C1P), which is
generated through direct phosphorylation of ceramide by ceramide
kinase (CerK) (2, 5, 6). Although CerK is the only enzyme de-
scribed for the generation of C1P so far, there is convincing evi-
dence for the existence of a CerK-independent metabolic pathway
(7). There are still detectable levels of C1P in CerK-deficient
animals (8) and in baculovirus-infected Sf9 cells treated with
CerK inhibitor (9). Notably, although C1P was identified .20 y
ago, some of its biological functions have only been revealed in
the last few years. C1P has been shown to act either as an intra-
cellular second messenger (10) or as an extracellular mediator
binding to a recently functionally identified, but still not cloned,
specific G protein–coupled receptor upon secretion to the extra-
cellular milieu. This specific C1P-coupled receptor was discov-
ered and described only on RAW 264.7 macrophages yet (7, 11). It
has been shown that C1P regulates cell proliferation and apoptosis
(1–3, 5–7, 10–12). In addition, there is increasing evidence that
C1P plays a role in inflammatory responses, because it stimulates
phagocytosis of neutrophils (8, 13), induces migration of macro-
phages (11, 14), as well as synthesis of eicosanoids (15), and
modulates cytokine production by macrophages and mast cells (1,
5, 16, 17). Initial studies demonstrated an increase of ceramide
concentration in apoptotic cells after stimulation of J774 macro-
phages with LPS (16). Moreover, an anti-inflammatory effect of
C1P was shown in LPS-stimulated HEK 293 cells and human
PBMCs (18).
Acute lung injury (ALI) (19) occurs in the setting of an acute
severe illness accompanied by systemic inflammation (20, 21) and
is characterized by diffuse parenchymal pulmonary inflammation
and edema (20–23). Intratracheal (i.t.) and i.p. instillation of LPS,
a bacterial cell wall component, to rodents is a well-accepted
and common experimental model for ALI from pulmonary and
extrapulmonary origin (24, 25) featuring key pathological com-
ponents of the disease such as profound neutrophilic lung re-
cruitment, vascular leakage/lung edema, and subsequent systemic
inflammation (20, 26). Unfortunately, despite marked efforts and
multiple therapeutic strategies, the mortality rate of patients suf-
fering from ALI remains high (21, 27–29).
In this study, we used an LPS-induced mouse model of ALI to
investigate the effect of C8-C1P and C16-C1P treatment in a
prophylactic and therapeutic setting. To adapt our observations to a
human situation, we treated LPS-stimulated human blood isolated
neutrophils with C1P in vitro. As a potential pathomechanistic link,
the modulation of the NF-kB pathway by C1P was studied.
*Department of Pneumology, COPD and Asthma Research Group, University
Hospital Freiburg, 79106 Freiburg, Germany;
†
Department of Biochemistry and
Molecular Biology, University of the Basque Country, 48080 Bilbao, Spain; and
‡
Organ Protection Program, Department of Anesthesiology, University of Colorado
School of Medicine, Aurora, CO 80045
ORCIDs: 0000-0001-7043-6692 (K.A.); 0000-0002-9964-6367 (N.E.).
Received for publication October 29, 2014. Accepted for publication December 14,
2015.
This study was supported by a grant from the German Research Foundation, ID 7/8–1
(to M.I.), National Institutes of Health Grants R01 DK097075, R01-HL0921, R01-
DK083385, R01-HL098294, and POIHL114457-01, and a grant from the Crohn’s
and Colitis Foundation of America (to H.K.E.).
Address correspondence and reprint requests to Dr. Marco Idzko, University
Hospital Freiburg, Department of Pneumology, COPD and Asthma Research
Group, Killianstrasse 5, 79106 Freiburg, Germany. E-mail address: marco.
idzko@uniklinik-freiburg.de
Abbreviations used in this article: ALI, acute lung injury; BALF, bronchoalveolar
lavage fluid; CerK, ceramide kinase; C1P, ceramide-1-phosphate; i.t., intratracheal;
KC, keratinocyte-derived chemokine; Mmp-9,matrix metalloproteinase 9; qPCR,
quantitative PCR; Traf2, TNFR-associated factor 2.
Copyright Ó2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402681
Published January 22, 2016, doi:10.4049/jimmunol.1402681
at Med Universitatsklinik Bibliothek on January 29, 2016http://www.jimmunol.org/Downloaded from
Materials and Methods
Reagents
Two types of C1P differing in carbonyl-chain length were used in our
experiments. Natural C16-C1P was purchased from Matreya LLC (Pleasant
Gap, PA) and solubilized in sterile nanopure water on ice using a probe
sonicator as previously described (14). Synthetic C8-C1P was acquired
from Cayman Chemical (Ann Arbor, MI) in a 1% solution of C8-C1P in
ethanol. The solution was aliquoted and frozen at 220˚C. Cells and mice
were not affected through the maximum concentration of 0.0005% ethanol
99%. Vehicle control means equally diluted in ethanol concentration in PBS
or medium in comparison with the diluted ethanol concentration of C8-C1P.
Animal studies
All experiments were approved by the local animal ethics committee and
conducted according to the Helsinki convention for the use and care of
animals. Male C57BL/6 mice (6–8 wk old) were bred at the animal facility
of the University Hospital Freiburg under specific pathogen-free condi-
tions. All animal experiments were performed using five animals per group
and were repeated three times.
LPS model of acute lung inflammation. First, mice were anesthetized using
ketamine (10 mg/kg; Intervet; Bela-Pharm GmbH, Vechta, Germany)/
rompun (150 mg/kg; Bayer, Leverkusen, Germany) solution injected i.p.
Second, they received an i.t. injection of LPS (300 mg/kg; Escherichia coli
Serotype 026:B6; Sigma-Aldrich GmbH, Steinheim, Germany) diluted in
50 ml sterile PBS. In addition, indicated concentration of C1P or vehicle
diluted in 80 ml sterile PBS was administered i.t. 1 h before (prophylactic
study) or 24 h after (therapeutic study) the LPS application. Mice were
euthanized 24 h after the last i.t. application.
Bronchoalveolar lavage fluid analysis by flow cytometry. Lungs were
washed three times through i.t. cannulae with 1 ml PBS containing 0.5 mM
EDTA. Total cell number was evaluated and differential cell distribution
was determined by flow cytometry (FACSCalibur; BD Biosciences, San
Diego, CA), by using the software CellQuest version 3.3 (BD Biosciences)
and FlowJo version 10 (Tree Star, Ashland, OR) as previously described
(30). In brief, mouse bronchoalveolar lavage fluid (BALF) cells were in-
cubated with unlabeled anti-CD16/CD32 to block Fc receptors and stained
for 20 min with anti-CD11c allophycocyanin, anti–Ly-6G (Gr-1) FITC,
anti-CD3e PE-Cy7, anti-CD45R (B220) PE-Cy7, and anti-mouse F4/80
PE (all from eBioscience, Frankfurt, Germany), in PBS containing 0.5%
BSA and 0.1% sodium azide. Gating strategy was as follows: first, non-
erythrocytes have been gated using forward scatter and side scatter. Next,
a lymphocyte gate was defined based on FCS and CD3
+
/B220
+
PE-Cy7
fluorescence. Neutrophils were determined as Gr-1
+
, CD3
2
/B220
2
cells,
whereas macrophages were defined as F4/80
+
, CD11c
+
, and CD3
2
/B220
2
population. The percentages obtained for each cell type were multiplied by
the number of total BALF.
Cytokine analysis. The levels of IL-1b, IL-6, IL-8, MIP-2, keratinocyte-
derived chemokine (KC), IFN-g, and TNF-awere measured in BALF and
neutrophil cell culture supernatants using ELISAs (Duoset; R&D Systems,
Minneapolis, MN), according to the manufacturer’s instructions. The de-
tection limit was 2 pg/ml. Samples with values below the detection limit
were assigned 1 pg/ml as cytokine concentration.
Plasma leakage assay. Plasma vascular leakage was examined as previously
described (31). In brief, Evans blue dye conjugated to albumin (20 mg/kg)
was injected into the tail vein of mice. Thirty minutes later, the mice were
sacrificed and the lungs were perfused with PBS supplemented with 5 mM
EDTA. Perfused lungs were excised en bloc, dried, weighed, and snap
frozen in liquid nitrogen. The whole lung was homogenized in PBS
(1 ml/100 mg tissue) before incubation in formamide at 60˚C for 18 h.
The OD of the supernatant was determined spectrophotometrically at
620 nm after centrifugation at 5000 3gfor 30 min. The concentration of
the extravasated Evans blue in lung homogenate was calculated against
the standard curve, and the results were expressed as microgram of
Evans blue dye per gram lung tissue (31).
Histology. Frozen lung tissue sections were cut and stained with H&E. The
density of neutrophils in the lung parenchyma was obtained as previously
described (32–35). In brief, 20 photomicrographs of lung parenchyma
(excluding areas of pulmonary vessels) were randomly obtained at 3400
magnification. The area of the whole photograph and the area of light (air
area) were calculated using the Software Image Pro Plus 4.0 (Media Cy-
bernetics, Rockville, MD). By subtracting the air area from the total
photograph area, we obtained the parenchyma tissue area. Then the
number of neutrophils was counted in the tissue area according to the
morphological criteria. The results were depicted as number of neutrophils
per square millimeter of parenchymal tissue (neutrophils/mm
2
).
Immunofluorescence and neutrophil quantification. Four-micrometer-thick
frozen lung sections were placed on polysine slides (Thermo Scientific), air-
dried, and fixed with cold acetone for 10 min. Tissue sections were washed
with PBS and blocked 1 h at room temperature with 5% goat serum, 1%
BSA, 0.1% cold fish skin gelatin (Sigma-Aldrich), 0.1% Triton X-100, and
0.05% Tween 20 in TBS. Sections were incubated with 1:1000 diluted GR-1
Ab (clone RB6-8C5; BioLegend) for 1 h at room temperature. As secondary
Ab, goat anti-rat Alexa555 (Life Technologies) was used. DAPI was
added during the last 10 min of secondary Ab incubation. Fluoromount
mounting medium (Sigma) and coverslips (36) were used to finish the
preparation. For imaging, Axioplan2 microscope with 633oil immer-
sion objective, AxioCam, and HAL100 have been used (all from Zeiss).
For image acquisition and analysis, Axiovision software v4.9.1.0 (Zeiss)
was used. Twelve high-power vision fields per lung were used for
counting neutrophils.
Total and phosphorylated NF-kB expression in lung tissue. Lungs were
homogenized in radioimmunoprecipitation assay buffer, containing PMSF,
natrium orthovanadate, phosphatase inhibitor mixture B, and protease in-
hibitor mixture (Santa Cruz Biotechnology, Santa Cruz, CA) on ice for
15 min and centrifuged at 17,000 3gat 4˚C for 15 min to remove the cell
debris. The amount of proteins was quantified by Quick Start Bradford
Protein Assay (Bio-Rad Lab GmbH, Munich, Germany). For all samples,
50 mg protein was loaded in NuPAGE 4–12% Bis-Trigel (Invitrogen AG,
Carlsbad, CA) and transferred to nitrocellulose membrane. The membrane
was blocked with 5% milk powder and incubated with a primary Ab
against phospho–NF-kB p65 (1:500, rabbit monoclonal IgG; Cell Signal-
ing, Danvers, MA) overnight. Afterward we used HRP-conjugated sec-
ondary Abs against rabbit IgG (Cell Signaling) for 1 h. Primary Ab was
visualized and enhanced by chemiluminescence with SuperSignal West
Dura (Thermo Scientific, Rockford, IL). The amount of mouse b-actin
detected by monoclonal anti-actin Ab clone C4 served as loading control
(MP Biomedicals LLC, Solon, OH). The densitometric analysis was per-
formed using ImageJ.
RNA isolation, cDNA synthesis, and quantitative PCR. Total RNA was
isolated with QIAzol lysis reagent for gene expression analysis (QIAGEN
GmbH, Hilden, Germany) following the manufacturer’s protocol. cDNA
synthesis was carried out using the First Strand cDNA synthesis kit
(Thermo Fisher Scientific GmbH, Schwerte, Germany). quantitative PCR
(qPCR) was performed on a LightCycler 480 (Roche Diagnostic GmbH,
Mannheim, Germany) using qPCR SYBR Green mix (Thermo Fisher
Scientific GmbH). b
2
-MICROGLOBULIN and GAPDH served as reference
genes. For all reactions the annealing temperature was 60˚C. Primer design
and relative quantifications were done as previously described (37); primer
sequences are available upon request.
In vitro studies
Human neutrophil isolation and culture. Human blood neutrophils were
obtained from venous blood using human Pancoll gradient (PAN-Biotech
GmbH, Aidenbach, Germany) as previously described (38). Isolated neu-
trophils were resuspended in PBS and their purity determined by using
Giemsa staining (.98%). Neutrophils were seeded in 24-well plates (2 3
10
6
/well) in RPMI 1640 supplemented with 10% FCS and penicillin/
streptomycin, and incubated at 37˚C with 5% CO
2
in a humidified at-
mosphere. Neutrophils were stimulated with C1P (1, 10 mM) or vehicle
for1hbeforeand1hafterLPS(1.5mg/ml) stimulation. Finally, su-
pernatant was collected 6 h after the last treatment and analyzed for IL-8
concentration.
NF-kB phosphorylation assay. Human neutrophils were seeded in six-well
plates (5 310
6
/well) in RPMI 1640 with 0.1% FCS and penicillin/
streptomycin. One hour before a 15-min LPS stimulation (1.5 mg/ml),
cells were preincubated with C1P 1 mM. Subsequently, collected cells
were wash ed in cold PBS a nd pr oteins wer e ext rac ted using radio-
immunoprecipitation assay lysis buffer as described earlier. Equal amounts
of proteins (20 mg) were analyzed for b-Actin and phospho–NF-kB p65
(Ser
536
) (rabbit monoclonal IgG; Cell Signaling).
Statistical analysis. If not stated otherwise, groups were compared using
one-way ANOVA, followed by Bonferroni comparison test (GraphPad
Prism 5 Software, San Diego, CA). The pvalues ,0.05 were regarded
as significant.
Results
Effects of C1P on LPS-induced ALI
i.t. instillation of LPS (300 mg/kg) in C57BL/6 for 24 or 48 h
resulted in severe acute lung inflammation, as demonstrated by
increased BALF cell numbers (neutrophils and macrophages),
2 C1P REDUCES LPS-INDUCED ACUTE LUNG INJURY
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elevated BALF-cytokine levels (Figs. 1, 2), plasma leakage into
the lungs (Fig. 3), and leukocyte infiltration in lung parenchyma
(Fig. 4). The prophylactic administration of both 1 and 10 mM
C1P, 1 h before LPS administration, resulted in lower numbers of
neutrophils and macrophages in BALF (Fig. 1A), decreased levels
of proinflammatory cytokines, notably KC and MIP-2 (Fig. 1B),
IL-6, and TNF-a(Fig. 1C), and significant reduction in micro-
vascular plasma leakage to the lung (Fig. 3A). This was accom-
panied by a decreased amount of neutrophils (Fig. 4A, 4E) and
inflammatory infiltrate in lung parenchyma (Fig. 4C).
Interestingly, there seems to be a dose response for C8-C1P, but
not for C16-C1P. Especially in the prophylactic LPS model, cy-
tokine levels of MIP-2 or IL-6 are more reduced by the treatment
of C8-C1P 10 mM instead of C8-C1P 1 mM. Administration of
C16-C1P independent of the concentration showed similar effects.
Notably, the beneficial effects of C1P on the cardinal features of
LPS-induced lung inflammation were also observed when animals
were treated with C1P 24 h after the administration of LPS-induced
ALI (Figs. 2, 3B). Histological examination of the lungs with H&E
and immunohistochemistry staining with Gr-1 for neutrophils from
mice treated with LPS for 48 h showed a significantly higher
number of neutrophils in the lung parenchyma than in the lungs of
C1P-treated mice (Fig. 4B, 4D, 4F), suggesting that C1P can not
only prevent but also treat established ALI. In addition, previous
experiments have shown that C1P reduced the early inflammatory
response after an LPS stimulation of 6 h (data not shown).
C1P reduced phosphorylation of NF-kB p65 expression in lung
tissue
LPS-induced lung injury has been reported to increase NF-kBac-
tivation in the lungs (39). Thus, we next questioned whether C1P
can decrease LPS-induced NF-kB activation in the prophylactic
and therapeutic settings of ALI. As shown in Fig. 5A, C16-C1P and
C8-C1P 1 mM significantly reduced LPS-induced NF-kB p65 ac-
tivation in the prophylactic model. Similar effects of C1P are shown
on LPS-induced phospho–NF-kB p65 in the therapeutic setting.
C1P attenuated mRNA NF-kB2 expression in lung tissue
Furthermore, qPCR analysis revealed that the protective role of C1P
can also be linked to the reduced mRNA expression of NF-kB2
(Fig. 6A, 6D) and NF-kBtarget genes including CD83 molecule
(CD83)andmatrix metalloproteinase 9 (Mmp-9) (Fig. 6A, 6D),
CCR5 and IL-6 (Fig. 6B, 6E), Myd88 (Fig. 6B), TNFR-associated
factor 2 (Traf2) (Fig. 6E), and Foxp3 (Fig. 6C, 6F). Myd88 plays a
key role in the innate and adaptive immune response and is included
in the activation of several proinflammatory genes. Especially
mRNA level of IL-6 was highly induced by LPS and attenuated
with prophylactic and/or therapeutic C1P treatment. Reduced IL-15
mRNA by C1P leads to lower STAT3 mRNA, which is involved in
many cellular processes such as cell growth and apoptosis (Fig. 6C,
6F). To conclude, C1P reduced the expression of LPS-induced
genes, which are involved in the regulation of NF-kB signaling.
Effects of C1P on LPS-induced NF-kB activation and IL-8
production in neutrophils
Neutrophil recruitment and activation play a pivotal role in the
pathophysiology of ALI; thus, we investigated the effects of C1P
on LPS-induced IL-8 secretion and NF-kB activation in purified
human blood neutrophils.
According to our in vivo study, we determined the prophylactic
and the therapeutic effect of C1P on the LPS-induced IL-8 pro-
duction. Thus, neutrophils were treated with C1P (1 or 10 mM)
either 1 h before or 1 h after LPS stimulation (1.5 mg/ml). Su-
pernatants were collected after an additional 7 h. As shown in
Fig. 7, C1P administration before (Fig. 7A) or after (Fig. 7B) LPS
stimulation significantly decreased LPS-induced IL-8 production.
For the latter, neutrophils (5 310
6
) were incubated with C1P or
vehicle 60 min before stimulation with LPS for an additional
15 min. Pretreatment of neutrophils with C1P before LPS pulsing
led to significant reduction in phospho–NF-kB p65, compared
with vehicle-treated, LPS-stimulated neutrophils, so that treatment
with C1P significantly reduced the NF-kB activation (Fig. 7C). In
Fig. 7D we present a representative Western blotting for phospho–
NF-kB and b-actin.
Discussion
Sphingolipids have been show n pr ev io usly to act a s pr oin-
flammatory or anti-inflammatory agents (18, 40). Although
the role of sphingosine-1-phosphate in LPS-induced lung inflam-
mation has been extensively studied (12, 31, 41), the influence of
FIGURE 1. Prophylactic administration of C1P (1 and 10 mM) before LPS administration significantly reduced ALI. C8-C1P (upper panels) and C16-C1P
(lower panels) (1 and 10 mM) were given 1 h before LPS administration and the animals were euthanized 24 h after LPS instillation. BALF cell differential
count was measured by flow cytometry (A). Concentration of KC, MIP-2 (B) and IL-6, TNF-ain BALF (C) were determined by ELISA. One representative
experiment out of three is shown. Values are given as mean 6SEM. n= 5 mice in each group. *p,0.05, **p,0.01, ***p,0.001 versus vehicle/LPS.
The Journal of Immunology 3
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C1P in this process is not well-known. Moreover, the exact
pathogenesis and molecular mechanisms leading to ALI (19) are
still poorly understood (20, 21, 42, 43). Hence specific therapies
have not been identified yet, and the current management for ALI
is mainly supportive (20, 21, 43). Our present study concentrates
on C1P as an anti-inflammatory modulator of LPS-induced acute
lung inflammation. A low-dose LPS model was used to reproduce
important biomarkers of ALI, such as edema formation and
proinflammatory cytokines release, according to the recommen-
dations from the American Thoracic Society (44).
In this article, we demonstrate for the first time, to our
knowledge, that both natural C16-C1P and the synthetic C8-C1P
analog attenuate LPS-induced ALI in mice. Intrapulmonary ap-
plication of C1P, before or after LPS administration, reduces the
amount of neutrophils and the production of proinflammatory
cytokines, and enhances the vascular leakage in the lung. Mech-
anistically we provide evidence that C1P inhibits LPS-induced
NF-kB2 mRNA expression and NF-kB activation in lung tissue
in vivo and neutrophils in vitro, and reduces LPS-primed IL-8
production by neutrophils.
ALI is characterized by pulmonary inflammation resulting from
microvascular endothelial barrier failure followed by a rich protein
pulmonary edema accumulation (20, 21, 26, 45, 46). Especially the
number of neutrophils seems to play a key role in the severity of
ALI (47). The intrapulmonary administration of LPS in rodents
has been accepted as a clinically relevant model of ALI (20, 26).
i.t. exposure of mice to LPS results in a massive recruitment of
neutrophils to the lungs as seen by increased BALF-neutrophilia at
24 and 48 h after LPS administration. The intrapulmonary treatment
of mice with C1P significantly reduces BALF-neutrophilia. Fur-
thermore, histological examination of the lungs from mice treated
with LPS and immunohistochemistry staining with Gr-1 display a
higher amount of neutrophils in the lung parenchyma in comparison
with mice that received C1P in a prophylactic or therapeutic setting.
In addition, LPS-induced proinflammatory cytokines, such as KC,
MIP-2, IL-6, and TNF-a, which actively participate in the patho-
genesis of ALI (e.g., by contributing to inflammatory cell recruit-
ment, activation, and migration) (20, 26, 48, 49), were attenuated by
C1P. Our findings were supported by Jo
´zefowski et al. (16), who
described C1P as a negative regulator of TNF-aproduction by LPS
in macrophages. Finally, IL-8 and its two homologs, KC and MIP-2
in mice, are important for the recruitment and activation of neu-
trophils (20, 26, 50) in ALI. IL-8 levels in BALF correlate with the
severity and prognosis of the disease (20, 50). Thus, our observation
that C1P inhibits these major cytokines involved in the induction
and maintenance of LPS-induced ALI underlines the potency of this
compound.
Nevertheless, differences are visible between C8-C1P concen-
trations, although C16-C1P independent of the concentration is
more stable in the effect. One explanation could be the natural
origin of the C16-C1P compound. Further pharmacokinetic and
pharmacodynamics studies could help to fully understand how
those active compounds were adsorbed and distributed in the body
to their capability to influence the immune system.
FIGURE 2. Therapeutic administration of C1P (1 and 10 mM) after LPS administration significantly reduced ALI. C8-C1P (upper panels) and C16-C1P
(lower panels) (1 and 10 mM) were given 24 h after LPS administration. Animals were euthanized 48 h after LPS instillation. BALF cell differential count
was analyzed by flow cytometry (A). KC, MIP-2 (B), IL-6, and TNF-awere determined in BALF by ELISA (C). One representative experiment of three is
shown. Values are given as mean 6SEM. n= 5 mice in each group. *p,0.05, **p,0.01, ***p,0.001 versus vehicle/LPS 48 h.
FIGURE 3. Prophylactic and therapeutic C1P treatment reduced LPS-induced plasma leakage. C1P (1 mM) was administered 1 h before LPS or 24 h
after LPS instillation. In the prophylactic study (A), animals were euthanized 24 h and in the therapeutic study (B) 48 h after LPS administration. Plasma
leakage was determined spectrophotometrically 18 h after Evans blue dye albumin (20 mg/kg) was injected into the tail vein. Values are given as mean 6
SEM. n= 5 mice in each group. The experiment was performed twice. *p,0.05, **p,0.01 versus vehicle/LPS 24 h or versus LPS 48 h/vehicle.
4 C1P REDUCES LPS-INDUCED ACUTE LUNG INJURY
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FIGURE 4. Effect of C1P on neutrophil recruitment in ALI. C8-C1P (upper panel) or C16-C1P treatment (lower panel) (1 and 10 mM) were given 1 h
before LPS or 24 h after LPS administration. For the prophylactic study (A), animals were euthanized 24 h after LPS and for therapeutic study 48 h after
LPS instillation (B). Histological pictures from stained lungs with H&E: prophylactic treatment (C) with C1P and therapeutic administration (D) of C1P.
Scale bars, 50 mm. Fluorescently labeled neutrophils in the lung parenchyma were counted: prophylactic treatment with C1P (1 mM) (E) and therapeutic
administration of C1P (1 mM) (F). Values are given as mean 6SEM. n= 5 mice in each group. *p,0.05, **p,0.01, ***p,0.001 versus vehicle/LPS
24 h or versus vehicle/LPS 48 h.
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Furthermore, the anti-inflammatory effect of C1P seems strongly
influenced by the used C1P concentration. In our previous study,
we already described only an anti-inflammatory effect until a C1P
concentration of 10 mM (51), which is in line with the results of
Hankins et al. (18). Often a proinflammatory effect of C1P (14,
52) is described either with higher C1P concentrations or different
solvent (53, 54). Further studies could clarify the full potential of
the C1P concentration regarding the interaction with the cells and
the role as exogenous or endogenous C1P compound.
A cardinal feature of LPS-induced ALI is the presence of
vascular leakage, leading to the development of pulmonary edema
(20, 26). A significant improvement in lung edema was observed
by pretreatment and posttreatment of animals with C1P. The re-
duction in lung edema might be related to the inhibition of neu-
trophil recruitment, because neutrophils are considered as the
primary cellular effectors of alveolar-capillary damage in ALI.
Notably, the potent anti-inflammatory capacity of C1P in ALI is
further supported by the fact that this effect was still observed in
mice with established LPS-induced lung inflammation.
The pathogenesis of ALI is complex and implies various signal
transduction processes. Particular attention has been given to the
NF-kB pathway, which regulates the expression of genes encoding
FIGURE 5. Prophylactic (A) and therapeutic
(C) C1P administration reduced NF-kB p65
protein expression and activation in lung tissue.
C1P (1 mM) was given before or 24 h after LPS
administration. Animals were euthanized 24 or
48 h after LPS administration, respectively. The
densitometric analysis of phospho–NF-kΒp65
was performed using ImageJ. Western blotting
analysis of the total extract (50 mg) using
phospho–NF-kB p65 Ab. Western blot of the
NF-kB p65 phosphorylation (upper blot)is
shown (Band D). The protein loading control
was monitored by staining the same membrane
with b-actin (lower blot)(Band D). The black
lines indicate where parts of the image were
joined. Values are given as mean 6SEM. n=5
mice in each group. **p,0.05, ***p,0.001
versus vehicle/LPS 24 h or versus vehicle/LPS
48 h.
FIGURE 6. C1P attenuated NF-kBmRNA expression and NF-kB–related genes in lung tissue of mice. The effect of prophylactic (A–C) and therapeutic
(D–F) C1P (10 mM) treatment in LPS-induced acute lung inflammation on NF-kB2 and NF-kBtarget genes are shown. qPCR was performed for NF-kB2,
MMP-9, and CD83 molecule (CD83)(Aand D), CCR5 and IL-6 (Band E), Myd88 (B) and Traf2 (E), IL-15,STAT3, and Foxp3 (Cand F). b
2
-MICRO-
GLOBULIN and GAPDH were used as reference genes. Values are mean 6SEM. Values are means 6SEMs of at least two biological replicates. n= 4–5
mice in each group of two technical replicates. *p,0.05, **p,0.01, ***p,0.001 versus vehicle/LPS 24 h or versus LPS 48 h/vehicle.
6 C1P REDUCES LPS-INDUCED ACUTE LUNG INJURY
at Med Universitatsklinik Bibliothek on January 29, 2016http://www.jimmunol.org/Downloaded from
mediators involved in inflammatory lung process (55, 56). In-
creased nuclear translocation of NF-kB in the lungs of patients
with ALI has been reported to correlate with disease severity and
outcome (19, 36, 55–57). Moreover, inhibition of NF-kB activa-
tion leads to reduction of acute lung inflammation in experimental
models of ALI (48, 56), pointing out the crucial role of the NF-kB
pathway in the pathogenesis of ALI. Furthermore, increased NF-
kB activation in neutrophils of ALI patients showed an important
correlation with impaired outcomes of the disease, particularly with
diminished time in the ventilator postincubation and increased sur-
vival in critically ill patients with ALI (56). Interestingly, we ob-
served that C1P was able to attenuate LPS-evoked NF-kB2 mRNA
expression in the lung tissue of animals. In addition, we observed an
inhibitory effect of C1P on NF-kB–related genes such as CCR5,
Mmp9,CD83,Myd88,Traf2 ,IL-6,IL-15,STAT3,andFoxp3,which
are implied in the inflammatory process. Moreover, Western blot
assays of NF-kB phosphorylation (phospho–NF-kB p65) encourage
our finding that C1P affects the LPS-induced NF-kBsignaling
pathway in the lung of mice. Similarly, Hankins et al. (18) have
shown the inhibitory effect of C1P (10 mM) on LPS-triggered NF-
kB activation and cytokine release in human embryonic kidney cells.
Contrary to LPS stimulation, others reported that higher concentra-
tion of C1P (20 mM) activates NF-kB in the alveolar rat macrophage
cell line NR8383 (52) and in resting cells of the murine macrophage
cell line J774A.1 (14). These findings support the hypothesis of
Hankins et al. that the inhibition of NF-kB by exogenous C1P is
dose dependent and specific to TLR4.
Furthermore, to better clarify the role of C1P, cell culture ex-
periments with primary human neutrophils were performed. C1P
was able to inhibit LPS-triggered NF-kB p65 phosphorylation in
the prophylactic and the therapeutic model, which was also asso-
ciated with reduction in LPS-induced IL-8 production by these cells.
Although the role of S1P receptors in the pathogenesis of in-
flammatory disorders has been extensively studied (2, 4, 12), the
knowledge about a possible receptor of C1P and its interaction is
hardly described. C1P might exert its anti-inflammatory capacity
by suppressing the activation of the NF-kB pathway. We support
the argument of Hankins et al. (7, 18, 58) that fluctuations in C1P
levels determine its proinflammatory or anti-inflammatory effects,
and further studies are required to clarify the interaction of C1P
with the plasma membrane or possible delineated receptors, as
well as the effect of C1P in specific cell types. C1P can act either
as an intracellular second messenger (10) or as an extracellular
mediator binding to functionally identified, but still not cloned,
specific G protein–coupled receptor upon secretion to the extra-
cellular milieu (11). Further studies could focus especially on this
subject to fully understand the interaction of C1P and its molec-
ular mechanism.
Therefore, we conclude that C1P acts as an important anti-
inflammatory agent in LPS-induced acute lung inflammation.
Major characteristic parameters of ALI, such as neutrophil acti-
vation, proinflammatory cytokines, and vascular plasma leakage
are diminished. Mechanistically we showed that C1P attenuates
NF-kB activation in human neutrophils and the expression of NF-
kB–related genes in the lungs of mice. Thus, C1P and its analogs
offer novel therapeutic targets for the treatment of ALI.
Acknowledgments
We thank Zsofia Lazar for critically revising the manuscript. Further thanks
go to Jessica Beckert for assistance in mice experiments and cell culture.
Disclosures
The authors have no financial conflicts of interest.
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